practitioner's corner

Practitioner’s Corner

PRACTITIONER’S CORNER

In recent years many studies have investigated the production of cytokines in the supernatants of cell cultures from patients with delayed hypersensitivity reactions to drugs as an in vitro tool for the diagnosis of reactions of this type. The detection of cells producing interferon (IFN) Ƣ using ELISPOT [1,2] or the quantiÀcation of IFN-Ƣ produced both at the intracellular level [3] and in the supernatants of cell cultures [4-6] are the diagnostic methods that have received most attention. However, results have been highly variable, with sensitivity rates ranging from 26% to 91% and speciÀcity rates ranging from 60% to 100%. We analyzed 15 patients with delayed allergic reactions to amoxicillin diagnosed by oral challenge and/or patch tests and/or intradermal tests with delayed reading (Table). An oral challenge was not performed in 1 patient (#13) for

In Vitro Production of Ag-SpeciÀc IFN-a in Patients With Delayed Hypersensitivity to Amoxicillin R Martínez-Aranguren,1 PM Gamboa, 2 E García-Lirio,2 MJ Goikoetxea,1 G Gastaminza,1 ML Sanz1 1 Department of Allergology and Clinical Immunology, Clinica Universidad de Navarra, Pamplona, Spain 2 Allergy Service, Basurto Hospital, Bilbao, Spain Key words: IFN-a. Delayed hypersensitivity reactions. Amoxicillin. In vitro test. Palabras clave: IFN-a. Reacciones retardadas de hipersensibilidad. Amoxicilina. Tests in vitro.

Table. Medical Profile and Diagnostic Tests of Patients With Delayed Hypersensitivity Reactions to Amoxicillin. In Vitro Results for Patients and Healthy Controls, With Interferon (IFN) a Production Expressed as the Stimulation Index Stimulation Indexa Patients

Age, y

Sex

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

19 46 38 65 35 65 48 12 4 37 66 50 47 64 72

F M F M F F F F F F F F F F F

Controls 1 2 3 4

33 26 55 30

M F F F

Reaction Type GCE GCE, Ed GCE Exf Der GCE, Ed Exf Der GCE GCE GCE MFE GCE GCE LV Exf Der EEM – – – –

Time Since Reaction, mo

Diagnostic Test

WB

PBMCs

PBMC+PHA (ELISA/CBA)

11 96 2 40 170 42 114 1 2 6 53 360 7 50 60

PT OC* OC+ LIST OC+ OC+ OC+ OC+ OC+ OC+ PT, LIST OC+ SB, 2 times OC+ EC

0.93 1.05 n.d. 0.82 4.74 3.06 8.22 2.22 1.10 0.57 1.15 n.d. n.d. n.d. n.d.

1.81 0.90 1.39 1.00 1.73 2.72 0.88 1.21 2.55 1.37 0.97 1.17 n.d. n.d. n.d.

n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.67/93.00 2.38/11.77 2.05/2.49 7.84/5.20 3.09/2.23

– – – –

OCOCOCOC-

1.29 1.02 1.00 2.00

1.00 2.05 2.38 3.96

0.82/1.21 28.19/1.62 0.59/0.05 n.d.

Abbreviations: Ed, edema; EEM, exudative erythema multiforme; Exf Der, exfoliative dermatitis; GCE, generalized cutaneous exanthema; MFE, multilocular fixed exanthema; LIST, late intracutaneous skin test; LV, leukocytoclastic vasculitis; n.d., not done; OC+, positive oral challenge test; OC-, negative oral challenge test; PBMC, peripheral blood mononuclear cell; PBMC+PHA, PBMC stimulated with phytohemagglutinin; PT, patch test; SB, skin biopsy; WB, whole blood. a Calculated as IFN-a quantified in the supernatants of the cell cultures stimulated in the presence of amoxicillin divided by that quantified in the supernatants of cell cultures without stimulation.

© 2013 Esmon Publicidad

J Investig Allergol Clin Immunol 2013; Vol. 23(2): 125-140

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ethical reasons, as leukocytoclastic vasculitis was detected on 2 occasions on the third day following administration of amoxicillin. All the patients provided informed consent before the collection of blood for the analysis of IFN-Ƣ production in response to amoxicillin. With the aim of Ànding a rapid and simple in vitro method, we analyzed the production of IFN-Ƣ following Ag-speciÀc stimulation in the supernatants of samples of whole blood (WB) ) (24 hours, 37º C, 5% CO2) and of cultures of mononuclear cells isolated from peripheral blood (PBMCs) (0.5 ⴒ 106 cells/mL in RPMI 1640 + 10% FCS, 72 hours, 37º C, 5% CO2) from 10 patients with delayed allergic reactions to amoxicillin (Table) and 4 healthy controls. We then analyzed, based on the method previously described by Halevy and Grossman [6], Ag-specific production of IFN-Ƣ in the supernatants of cultures of PBMCs stimulated under suboptimal conditions with phytohemagglutinin A (PHA) (0.5 ⴒ 106 cells/mL in RPMI 1640 + 10% FCS + 5 +g/mL PHA, 72 hours, 37º C, 5% CO2) from 5 patients with delayed allergic reactions to amoxicillin and 3 healthy controls. Cells were cultured in duplicate and stimulated with 3 different concentrations of amoxicillin (0.5, 0.2, and 0.1 mg/mL); 10 +g/mL of PHA was used as a positive control and basal conditions (without amoxicillin) served as a negative control. The IFN-Ƣ in the supernatants of the cell cultures was quantiÀed using 2 techniques in parallel: enzyme-linked immunosorbent assay (ELISA) (Human IFN-Ƣ Opt EIA, BD Bioscience), and Áow cytometry (Cytometric Bead Array [CBA] Human Th1/Th2/ Th17 Cytokine Kit; BD Bioscience). Production of IFN-Ƣ was calculated using a stimulation index (SI) (IFN-Ƣ quantiÀed in the supernatants of the cell cultures stimulated in the presence of amoxicillin divided by that quantiÀed in the supernatants of cell cultures without stimulation). Sensitivity, speciÀcity, area under the curve (AUC), and the optimal cutoff for sensitivity and speciÀcity were estimated using receiver operator curves. Sensitivity and speciÀcity values were calculated for each of the 3 types of cultures (WB, PBMCs, and PBMCs+PHA). The Table summarizes the results obtained. The optimal cutoff was that where the SI was greater than 2 for the 3 types of culture. When IFN-Ƣ was quantiÀed using ELISA, the AUC for the SI was 0.55 for the WB cultures, 0.26 for the PBMC cultures, and 0.60 for the PBMC+PHA cultures. However, when quantiÀcation was performed using CBA, the AUC was 1. The main objective of this study was to test the quantiÀcation of IFN-Ƣ in the supernatants of WB stimulated with a drug for 24 hours as a rapid and simple method for the in vitro diagnosis of delayed hypersensitivity reactions to drugs. This technique yielded a speciÀcity of 100% but sensitivity was low (40%). Similar sensitivity values have been reported for PBMCs cultured for 72 hours [3,7] but higher values, in the range of 80% to 100%, have also been reported [4,5]. Our results for the PBMC cultures were worse in terms of both sensitivity (17%) and speciÀcity (25%). The best diagnostic values were obtained with the quantiÀcation of IFN-Ƣ secreted into the medium of PBMC cultures stimulated under suboptimal conditions with 5 +/mL of PHA; this is similar to the method used by Halevy and Grossman [6], although they used a larger quantity of PHA (200 +g/mL). When we quantiÀed IFN-Ƣ using ELISA, we obtained a speciÀcity of 67% and a sensitivity of 80%;

J Investig Allergol Clin Immunol 2013; Vol. 23(2): 125-140

CBA, however, yielded a speciÀcity and sensitivity of 100%. The different results can be explained by the small amount of IFN-Ƣ quantiÀed in the supernatants of the cell cultures from patient 11 and control 2 at baseline using CBA and ELISA, respectively. We conclude that the culture method providing the best results for the diagnosis of delayed hypersensitivity reactions to amoxicillin is quantiÀcation of IFN-Ƣ production following Ag-speciÀc stimulation for 72 hours using CBA in cultures of PBMCs stimulated in suboptimal conditions with 5 +g/mL of PHA. Further studies are needed to analyze the conditions assayed in our study with a larger group of patients.

Acknowledgments We thank the Foundation of the Spanish Society of Allergology and Clinical Immunology (SEAIC) for funding this research through one of their Research Aid scholarships.

References 1. Rozieres A, Hennino A, Rodet K, Gutowski M. C, Gunera-Saad N, Berard F, Hennino A, Nicolas JF. Detection and quantification of drug-specific T cells in penicillin allergy. Allergy. 2009; 64: 534-42. 2. Beeler A, Engler O, Gerber B O, Pichler W. Long-lasting reactivity and high frecuency of drug-specific T cells after severe systemic drug hypersensitivity reactions. J Allergy Clin Immunol. 2006;117(2):455-62. 3. Martin M, Wurpts G, Ott H, Baron JM, Erdmann S, Merk HF, Sachs B. In vitro detection and characterization of drug hypersensitivity using flow cytometry. Allergy. 2010;65:32-9. 4. Lochmatter P, Beeler A, Kawabata TT, Gerber BO, Pichler WJ. Drug specific in vitro release of IL-2, IL-5, IL-13 and IFN-Ƣ in patients with delayed-type drug hypersensibility. Allergy. 2009;64(9):1269-78. 5. Khalil G, El-Sabban M, Al Ghadban S, Azzi S, Shamra S, Khalifé S, Maroun R. Cytokine expression profile of sensitized human T lymphocytes following in vitro stimulation with amoxicillin. Eur. Cytokine Netw. 2008; 19: 131-41. 6. Halevy S, Grossman N. Multiple drug allergy in patients with cutaneous adverse drug reactions diagnosed by in vitro drug induced interferon-a release. Isr Med Assoc J. 2008;10:865-8. 7. Sachs B, Erdmann S, Malte Baron J, Neis M, al Masaoudi T, Merk HF. Determination of interleukin-5 secretion from drug-specific activated ex vivo peripheral blood mononuclear cells as a test system for the in vitro detection of drug sensitization. Clin Exp Allergy. 2002 May;32(5):736-44.

❚ Manuscript received August 20, 2012; accepted for publication, September 3, 2012.

María L. Sanz Departamento de Alergología e Inmunología Clínica Universidad de Navarra Pío XII 36 31008 Pamplona, Spain E-mail: [email protected]



Baseline Tryptase Levels Are Related to Age, Total IgE, and Anti-rPru p 3 IgE Levels in Peach-Allergic Patients EA Pastorello, 1 L Farioli, 1 G Scibilia, 1 V Pravettoni, 2 A Mascheri,1 C Stafylaraki,1 M Nichelatti,3 L Balossi,1 R Asero4 1 Allergology and Immunology Unit, Niguarda Ca’ Granda Hospital, Milano, Italy 2 Allergology and Immunology Unit, Foundation Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, IRCCS, Milano, Italy 3 Service of Biostatistics, Niguarda Ca’ Granda Hospital, Milan, Italy 4 Allergy Unit, Clinica San Carlo, Paderno Dugnano, Milan, Italy Key words: Tryptase. Lipid transfer protein. Food allergy. Palabras clave: Triptasa. Proteína transportadora de lípidos. Alergia alimentaria.

Tryptase and histamine are preformed mediators released by mast cells upon activation. Since tryptase persists for hours after an anaphylactic event, an increase in tryptase levels is currently considered the best available marker of anaphylaxis. Mast cells also constitutively secrete ß-tryptase, the serum levels of which reflect mast-cell burden. Accordingly, high levels of ß-tryptase suggest the presence of systemic mastocytosis. Moreover, moderately increased levels of tryptase associated with urticaria, Áushing, headache, and/ or gastrointestinal reactions in the absence of World Allergy Organization mastocytosis criteria were recently classiÀed as mast cell activation syndrome (MCAS). In allergy, apart from acute reactions, high tryptase levels have been reported only in hymenoptera venom allergy, in association with an increased risk of severe anaphylaxis from both stings and venom immunotherapy [1]. Hymenoptera venom allergy is also associated with mastocytosis [2]. Whether elevated baseline serum tryptase levels might also be a risk factor for severe reactions in food allergy is unknown. Lipid transfer protein (LTP), a plant panallergen [3], is the major cause of primary food allergy and food-induced anaphylaxis in Mediterranean countries [4,5]. The clinical expression of LTP hypersensitivity shows much variability, ranging from the total absence of symptoms to oral allergy syndrome (OAS), isolated gastrointestinal symptoms, urticaria/angioedema, and even severe anaphylaxis. The cause of such variability is unclear and only partially explained by LTP-speciÀc immunoglobulin (Ig) E levels [6]. Nonetheless, an association between high levels of anti-Pru p 3 (LTP) IgE and peach allergy severity may exist and cosensitization to Pru p 1 seems to attenuate clinical reactivity [7]. In this study we measured baseline serum tryptase levels in peach-allergic individuals and looked for a possible association with clinical variables (symptom severity, sex, age) and immunological variables (total IgE and Pru p 3 IgE levels).


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We studied 148 peach-allergic adults (41 males/107 females [P